Neuropeptide Y Y1 receptor association with beta-arrestin measured by bimolecular fluorescence complementation

Lead Research Organisation: University of Nottingham
Department Name: School of Life Sciences

Abstract

We are developing a new way to measure protein interactions involved in signalling from cell surface receptors, using powerful fluorescent microscopy techniques.

The receptor proteins are called G protein coupled receptors, present in every cell in the body, which recognise chemical neurotransmitters and hormones. We will focus on their association with important partners called arrestins. Arrestins regulate receptor activity, contributing to drug tolerance, and also organise signalling leading to cell survival or differentiation.

In our technique we add two halves of a fluorescent protein to the receptor and arrestin molecules, so that the light from it will only be seen when the segments are brought together by a receptor-arrestin complex. We will then identify these complexes by a range of imaging methods, some of which can examine behaviour at a single molecule level. This will tell us more about the molecular controls that govern the formation of different receptor-arrestin combinations, and how these controls generate alternative patterns of signalling.

We hope that this will help a wide scientific community, including those interested in the particular receptors under study (for the neurotransmitter Neuropeptide Y) as targets to treat obesity, and those who see potential for receptor drugs that selectively alter arrestin signalling.

Technical Summary

Beta-arrestins bind G protein coupled receptors (GPCRs) to initiate their desensitization and internalization, and are also signalling adaptors for protein kinase cascades. Efficient interaction with GPCRs involves recognition of activated and phosphorylated GPCR domains by two modular sensors in the arrestin protein. However it is now apparent that the molecular nature of the GPCR-arrestin complex varies with ligand or arrestin isoform, and that this variation is functionally important. For example, there is evidence for some GPCRs that antagonists can stimulate certain signalling cascades via arrestins but independently of G protein activation. In this application the technique of bimolecular fluorescence complementation (BiFC) will be used to evaluate at the single living cell level the nature of the interaction between the neuropeptide Y Y1 (NPY1) receptor and beta-arrestins by different ligands. In this technique, the association of NPY1 receptors with beta-arrestins can be followed by the complementation of fragments of either yellow or cyan fluorescent proteins (YFP / CFP; which themselves are non fluorescent) attached to the protein partners to regenerate YFP or CFP fluorescence. This will then be detected and quantitified in living cells using both confocal microscopy and (for detailed pharmacological analysis) a confocal plate reader. Using these techniques we will determine whether agonists (endogenous peptides e.g. neuropeptide Y and synthetic ligands) can direct association with specific arrestin isoforms, and whether Y1 receptor antagonists (of G protein coupled responses) can stimulate arrestin binding (!?dual efficacy!?). Arrestin binding determinants used by these different complexes will be determined by mutagenesis of the Y1 receptor activation and phosphorylation sites. In addition we will investigate whether GPCRs and arrestins interact as dimers. Finally, we will use
BiFC fluorescence complementation in association with fluorescence correlation spectroscopy to monitor the diffusional characteristics of individual Y1 receptor-arrestin complexes in membrane microdomains (e.g. clathrin coated pits) of single living cells.
Findings from this research will provide new insight into the mechanism of GPCR-arrestin interaction, and into signalling from the neuropeptide Y1 receptor which has widespread physiological roles and therapeutic potential.

Publications

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Sivertsen B (2011) Unique interaction pattern for a functionally biased ghrelin receptor agonist. in The Journal of biological chemistry

 
Description AJ Clark prize PhD studentship
Amount £75,680 (GBP)
Organisation British Pharmacological Society (BPS) 
Sector Learned Society
Country United Kingdom
Start 10/2010 
End 09/2013
 
Description Astra Zeneca, Alderley Park - ghrelin receptor / FFARs 
Organisation AstraZeneca
Country United Kingdom 
Sector Private 
PI Contribution We showed that the automated BiFC imaging assay for the Y1 receptor / beta-arrestin2 interaction, can be applied to the ghrelin receptor and free fatty acid receptors.
Collaborator Contribution AZ were interested in using our BiFC assay to screen ligands for FFAR and ghrelin receptors (see 2012 pulication on this). The availability of this technique was one element for securing industrial funding for an FFAR based PhD studentship (£40 k)
Impact Two of us (NDH/ SJH) secured a PhD studentship (2009 - 2013) with AZ consumables funding (£40 k) examining free fatty acid receptor function. Although the primary purpose of this studentship did not involve BiFC, our work in extending the BiFC arrestin assay to FFARs helped secured this funding.
Start Year 2008
 
Description NovoNordisk - consultancy on high content imaging 
Organisation Novo Nordisk
Country Denmark 
Sector Public 
PI Contribution consultancy with NovoNordisk to advise on high content imaging of G protein coupled receptors and the insulin receptor, including application of BiFC technology. Co-investigator on a NovoNordisk funded postdoctoral project at the Hagedorn Research Institute (Copenhagen), investigating insulin receptor trafficking.
Collaborator Contribution Named investigator on a postdoctoral project at NovoNordisk.
Impact None so far
Start Year 2010
 
Description University Community Day 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact NDH organised the Biomedical Sciences contributions to the University of Nottingham's Day of Exploration for the general public yearly in April, 2009 - 2011 and 2014, and participated in 2015. This included posters, microscope demonstrations and talks highlighting our research, including this grant. The event was open to the general public and our displays attracted 100-200 visitors in all years

The demonstrations proved particularly popular with school children (age 8 - 16). University run feedback from the visitor questionnaire was highly positive.
Year(s) Of Engagement Activity 2009,2010,2011,2014,2015
URL http://www.nottingham.ac.uk/mayfest/index.aspx
 
Description Wellcome Trust Small Arts Award - Jo Berry 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact I was co-investigator on a Wellcome Trust small Arts award to the artist Jo Berry (2010 - 2011, £28 k). Jo has carried out a scientific research project in our group to explore our use of imaging techniques in pharmacology, set up with the MRC grant. She has generated digital artwork based on our receptor images, and exhbited these works alongside scientific displays with the aim to communicate the science about drugs and receptors in an accesible format to the general public. School visits took place in 2011, with exhibitions at the Derby Art Gallery and Hospital in 2011, at the Lakeside Gallery Nottingham (2012), and at St.Paul's Gallery, Barnes, London (2012).

A report (2012) by the independent evaluator for this award is available on request. For example, 20 000 people visited the Derby Art Gallery during the exhibition; the work also received national media coverage (e.g. Daily Telegraph website) in addition to articles in broad reach scientific journals (e.g. BMJ) and sponsorship from the microscope manufacturer Zeiss.
Year(s) Of Engagement Activity 2010,2011,2012
URL https://www.youtube.com/watch?v=XTPnuL_gZV4